The invention was based on the object of finding novel compounds having valuable properties, in particular those which can be used for the preparation of medicaments.
The present invention relates to compounds and to the use of compounds for the treatment of diseases which are associated with an increase in the sphingosine phosphate level, furthermore to pharmaceutical compositions which comprise these compounds.
In detail, the present invention relates to compounds of the formula I, which preferably inhibit the enzyme sphingosine kinase 1, which regulates the sphingosine phosphate level by phosphorylation of sphingosine, to compositions which comprise these compounds, and to methods for the use thereof for the treatment of diseases and complaints, such as cancer, tumour formation, growth and spread, arteriosclerosis, eye diseases, choroidal neovascularisation and diabetic retinopathy, inflammatory diseases, arthritis, neurodegeneration, restenosis, heart diseases, wound healing or transplant rejection. In particular, the compounds according to the invention are suitable for the therapy of cancer diseases.
Sphingosine phosphate belongs to the molecule family of the sphingolipids, which, besides their role as structural building blocks of cell membranes, also exert important functions as extra- and intracellular signal molecules. Sphingosine phosphate (S1P) is formed in the cell from sphingomyelin, which initially breaks down to form ceramide and sphingosine, and the latter is phosphorylated by sphingosine kinases. Of the two sphingosine kinases identified to date, sphingosine kinase 1 (SphK1) is ascribed the greater importance in the formation of S1P in the serum (Zemann et al., 2006 Blood Vol 107 page 1454). While ceramide and sphingosine induce cell death and cell growth inhibition (Kolesnick 2002, J Clin Invest Vol 110, page 3; Ogretmen et al. 2004 Nat Rev Cancer Vol 4, page 604), sphingosine phosphate has an opposite effect on the cell and increases the resistance to apoptosis, cell growth and the discharge of messenger substances, which promote perfusion of the tissue and thus also of tumours (Cuvilier et al. 1996, Nature Vol 381, page 800; Perez et al. 1997, Nat Med Vol 3, page 1228). The ratio of ceramide and sphingosine on the one hand and S1P on the other is consequently decisive for cell growth, and inhibition of SphK 1 can thus not only suppress the formation of the growth-promoting sphingosine phosphate, but also increase the cellular concentration of the growth-inhibiting molecules ceramide and sphingosine.
A multiplicity of cellular effects which are triggered by S1P is promoted by secretion of S1P and binding thereof to to date 5 different G-protein-coupled receptors (known as S1P1-5). Signal propagation in turn takes place via various G-proteins (Gi, Gq, G12/13), meaning that a number of different cellular signailing pathways, such as, for example, ERK or PI3K, which are particularly important in cancer formation and growth, are activated. In addition, an increasing number of publications shows that S1P is an important factor in tumoral angiogenesis. Angiogenesis is an important process in tumour growth, by means of which blood vessels are re-formed starting from already existing ones and the supply of the tumour with nutrients is thus ensured. For this reason, inhibition of angiogenesis is an important starting point for cancer and tumour therapy. (Folkman, 2007, Nature Reviews Drug Discovery Vol. 6, page 273-286). S1P stimulates chemotactic movement of endothelial cells and induces differentiation to give multicellular structures, both early steps in the formation of new blood vessels (Lee et al. 1999 Biochem Biophys Res Commun Vol 264 page 325; Argraves et al. 2004 J Biol Chem Vol 279 page 50580). In addition, S1P promotes the migration of endothelial precursor cells originating from bone marrow to neovascular initiation sites (Annabi et al. 2003 Exp Hematology Vol 31 page 640) and transactivates the receptor of VEGF, one of the most important proangiogenic factors, in particular in tumour biology (Tanimoto et al. 2002 J Biol Chem Vol 277 page 42997; Endo et al. 2002 J Biol Chem Vol 277 page 23747). Direct evidence of the activity of S1P in tumour angiogenesis has been provided by experiments with an antibody which binds specifically to S1P. The S1P antibody inhibited the migration and vascularisation of endothelial cells in vitro, blocked the S1P-dependent secretion of proangiogenic factors, such as VEGF, IL-8 and IL-6, in vitro and in vivo and significantly reduced the growth of tumour models of the breast, lung and ovaries in mouse xenograft experiments (Visentin 2006 Cancer Cell Vol 9 page 225).
In addition, S1P also has intracellular functions, such as, for example, the activation of the transcription factor NF-κB, which plays a major role in apoptosis resistance of cancer cells (Xia et al. 2002 J Biol Chem Vol 277 page 7996). However, the intracellular interaction partners of S1P have not yet been identified.
It follows from this that, in contrast to a likewise conceivable intervention with the cancer-promoting action of S1P by pharmacological blockade of the extracellular receptors, inhibition of the enzyme SphK1, which is responsible for S1P formation, has the advantage of thus also suppressing the intracellular activities of S1P. This approach is supported by investigations by Xia et al. (2000 Curr Biol Vol 10 page 1527), which show that non-tumorigenic fibroblasts are transformed by ectopic expression of SphK1 and can form tumours in mice. SphK1 can thus be classified as an oncogene. In various expression studies, increased SphK1-mRNA concentrations in tumour tissues of the brain, breast, lung, ovaries, stomach, uterus, kidneys and small and large intestine have been observed compared with healthy tissue (French et al. 2003 Cancer Research Vol. 63 page 5962; Johnson et al. 2005 J Histochem Cytochem Vol 53 page 1159; Van Brocklyn et al. 2005 J Neuropathol Exp Neurol Vol 64 page 695). In addition, increased expression of SphK1 correlates with a worse prognosis in patients with glioblastoma multiforme (Van Brocklyn et al. 2005 J Neuropathol Exp Neurol Vol 64 page 695). SphK1 has an important role in the modulation of the apoptosis of cancer cells induced by chemotherapeutic agents. Thus, overexpression of SphK1 increases the resistance of breast cancer, prostate cancer and leukaemia cells to chemotherapeutic agents, such as anthracyclines, docetaxel, camptothecin or doxorubicin (Nava et al. 2002 Exp Cell Res Vol 281 page 115; Pchejetski 2005 Cancer Res Vol 65 page 11667; Bonhoure 2006 Leukemia Vol 20 page 95). It has been shown that the increased presence of SphK1 results in a shift in the ceramide/S1P equilibrium towards S1P, which promotes apoptosis resistance. A possible mechanism here is the inhibition of the mitochondrial cytochrome C discharge by SphK1, which normally represents an early event in programmed cell death (Cuvilier et al. 2001 Blood Vol 98 page 2828; Bonhoure 2006 Leukemia Vol 20 page 95).
Conversely, specific blockade of SphK1 expression by means of siRNA in tumour cell models of various indications, such as leukaemia, breast cancer, glioblastoma or prostate cancer, enables apoptosis to be triggered or the effect of chemotherapeutic agents to be increased (Bonhoure 2006 Leukemia Vol 20 page 95; Taha et al. 2004 J Biol Chem Vol 279 page 20546; Taha et al. 2006 FASEB J Vol 20 page 482; Van Brocklyn et al. 2005 J Neuropathol Exp Neurol Vol 64 page 695; Pchejetski 2005 Cancer Res Vol 65 page 11667).
It has been shown in a mouse model that overexpression of SphK1 triggers degenerative changes of cardiomyocytes and myocardial fibrosis, which increased with increasing age of the experimental animals. A function of the S1P signalling pathway in heart diseases is also supported by the fact that the formation of cardiovascular fibroses is strongly inhibited in mice in which the expression of the S1P3 receptor has been specifically suppressed (Takuwa 2008 Biochimica and Biophysica Acta in press). S1P also has a role in the differentiation of fibroblasts to give myofibroblasts and thus in the formation and progression of fibrotic diseases in other organs, such as, for example, the lung (Kono et al. 2007 Am J Respir Cell Mol Biol page 395).
It has been found that the compounds according to the invention cause specific inhibition of sphingosine kinase 1, but not of sphingosine kinase 2. The compounds according to the invention preferably exhibit an advantageous biological activity which can be detected in the tests described herein, for example. In such tests, the compounds according to the invention exhibit and cause an inhibiting effect, which is usually documented by IC50 values in a suitable range, preferably in the micromolar range and more preferably in the nanomolar range.
In general, all solid and non-solid tumours can be treated with the compounds of the formula I, such as, for example, monocytic leukaemia, brain, urogenital, lymph system, stomach, laryngeal, ovarian and lung carcinoma, including lung adenocarcinoma and small-cell lung carcinoma. Further examples include prostate, pancreatic and breast carcinoma.
As discussed herein, effects of the compound according to the invention are relevant for various diseases. Accordingly, the compounds according to the invention are useful in the prophylaxis and/or treatment of diseases which are influenced by inhibition of SphK1.
The present invention therefore relates to compounds according to the invention as medicaments and/or medicament active ingredients in the treatment and/or prophylaxis of the said diseases and to the use of compounds according to the invention for the preparation of a pharmaceutical agent for the treatment and/or prophylaxis of the said diseases, and also to a method for the treatment of the said diseases comprising the administration of one or more compounds according to the invention to a patient in need of such an administration.
The host or patient can belong to any mammalian species, for example a primate species, particularly humans; rodents, including mice, rats and hamsters, rabbits, horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, where they represent a model for the treatment of human disease.
The sensitivity of a particular cell to treatment with the compounds according to the invention can be determined by in vitro tests. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period of time which is sufficient to enable the active agents to lower the intracellular S1P concentration and in addition to block the secretion of angiogenesis-promoting substances or to induce cell death. For testing in vitro, cultivated cells from a biopsy sample or established cancer cell lines in which SphK1 is overexpressed can be used.
The dose varies depending on the specific compound used, the specific disease, the patient status, etc. A therapeutic dose is typically sufficient to considerably reduce the undesired cell population in the target tissue, while the viability of the patient is maintained. The treatment is generally continued until a considerable reduction has occurred, for example at least about 50% reduction in the cell burden, and can be continued until essentially no undesired cells can be detected in the body.
Use
As described in the introduction, SphK1, S1P and the cell surface receptors S1P1-5 thereof are involved in a multiplicity of physiological and pathophysiological processes. For this reason, it can be expected that the inhibition of SphK1 by the substances described here can be utilised for therapeutic purposes in various diseases.
The formation of S1P by SphK1 and the associated shift in the ceramide/S1P equilibrium results, as stated above, in the cells proliferating to a greater extent and becoming more resistant to apoptotic stimuli. A general function of SphK1 can be derived therefrom in hyperproliferative diseases, such as cancer, psoriasis, restenosis and arteriosclerosis. The compounds of the formula I on which this invention is based and which inhibit SphK1 and thus regulate and/or modulate the S1P level, compositions which comprise these compounds, and the methods described can thus be employed for the treatment of these diseases. In general, all solid and non-solid tumours can be treated with the compounds of the formula X, such as, for example, monocytic leukaemia, brain, urogenital, lymph system, stomach, laryngeal ovarian and lung carcinoma, including lung adenocarcinoma and small-cell lung carcinoma. Further examples include prostate, pancreatic and breast carcinoma.
Besides the function in cell growth, S1P also plays a role in the neoformation of blood vessels (angiogenesis). In many disease processes, angiogenesis is either causally at the centre of the disease or has a worsening effect on the progression of the disease. In cancer events, for example, angiogenesis results in the tumour being enlarging and possibly spreading into other organs. Further diseases in which angiogenesis plays an important role are psoriasis, arthrosis, arteriosclerosis and eye diseases, such as diabetic retinopathy, age-induced macular degeneration, rubeosis iridis or neovascular glaucoma. The compounds of the formula I on which this invention is based and which inhibit SphK1 and thus regulate and/or modulate the S1P level, compositions which comprise these compounds, and the methods described can thus be employed for the treatment of these diseases.
Furthermore, SphK1 and S1P influence the proliferation, differentiation, migration and secretion of immune cells (Rosen and Goetzl 2005 Nat Rev Immunol Vol 5 page 560) and are thus involved in various functions of the immune system and in inflammatory processes. Stimulation of the immune system increases the formation and discharge of S1P in mast cells, blood platelet cells and some mononuclear phagocytes (Stunff et al. 2004 J Cell Biochem Vol 92 page 882; Olivera and Rivera 2005 j Immunol Vol 174 page 1153). The activity of SphK1 is greatly increased, in particular, by factors such as tumour necrosis factor (TNF) and crosslinking of IgG receptors (Stunff et al. 2004 J Cell Biochem Vol 92 page 882; Delon et al. 2004 J Biol Chem Vol 279 page 44763). In addition, it has been shown that SphK1 and S1P are important for the TNF-dependent formation of pro-inflammatory enzymes, such as cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) (Pettus et al. 2003 FASEB J Vol 17 page 1411; Kwon et al. 2001 J Biol Chem Vol 276 page 10627-33). The compounds of the formula I on which this invention is based and which inhibit SphK1 and thus regulate and/or modulate the S1P level, compositions which comprise these compounds, and the methods described can thus be employed for the treatment of inflammation-induced diseases, such as arthrosis, arteriosclerosis, psoriasis, multiple sclerosis, chronic inflammatory bowel diseases (Crohn's disease, colitis ulcerosa) asthma and other allergic diseases.
The compounds of the formula I can furthermore be used for the isolation and investigation of the activity or expression of Sph kinase. In addition, they are particularly suitable for use in diagnostic methods for diseases in connection with unregulated or disturbed Sph kinase activity.
It can be shown that the compounds according to the invention have an antiproliferative action in vivo in a xenotransplant tumour model. The compounds according to the invention are administered to a patient having a hyperproliferative disease, for example to inhibit tumour growth, to reduce inflammation associated with a lymphoproliferative disease, to inhibit trans-plant rejection or neurological damage due to tissue repair, etc. The present compounds are suitable for prophylactic or therapeutic purposes. As used herein, the term “treatment” is used to refer to both prevention of diseases and treatment of pre-existing conditions. The prevention of proliferation is achieved by administration of the compounds according to the invention prior to the development of overt disease, for example to prevent the growth of tumours, prevent metastatic growth, diminish restenosis associated with cardiovascular surgery, etc. Alternatively, the compounds are used for the treatment of ongoing diseases by stabilising or improving the clinical symptoms of the patient.
The host or patient can belong to any mammalian species, for example a primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
The susceptibility of a particular cell to treatment with the compounds according to the invention can be determined by in vitro tests. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period of time which is sufficient to allow the active agents to induce cell death or to inhibit migration, usually between about one hour and one week. In vitro testing can be carried out using cultivated cells from a biopsy sample. The viable cells remaining after the treatment are then counted.
The dose varies depending on the specific compound used, the specific disease, the patient status, etc. A therapeutic dose is typically sufficient considerably to reduce the undesired cell population in the target tissue while the viability of the patient is maintained. The treatment is generally continued until a considerable reduction has occurred, for example an at least about 50% reduction in the cell burden, and may be continued until essentially no more undesired cells are detected in the body.
For identification of a signal transduction pathway and for detection of interactions between various signal transduction pathways, various scientists have developed suitable models or model systems, for example cell culture models (for example Khwaja et al., EMBO, 1997, 16, 2783-93) and models of transgenic animals (for example White et al., Oncogene, 2001, 20, 7064-7072). For the determination of certain stages in the signal transduction cascade, interacting compounds can be utilised in order to modulate the signal (for example Stephens et al., Biochemical J., 2000, 351, 95-105). The compounds according to the invention can also be used as reagents for testing kinase-dependent signal transduction pathways in animals and/or cell culture models or in the clinical diseases mentioned in this application.
For identification of a signal transduction pathway and for detection of interactions between various signal transduction pathways, various scientists have developed suitable models or model systems, for example cell culture models (for example Khwaja et al., EMBO, 1997, 16, 2783-93) and models of transgenic animals (for example White et al., Oncogene, 2001, 20, 7064-7072). For the determination of certain stages in the signal transduction cascade, interacting compounds can be utilised in order to modulate the signal (for example Stephens et al., Biochemical J., 2000, 351, 95-105). The compounds according to the invention can also be used as reagents for testing kinase-dependent signal transduction pathways in animals and/or cell culture models or in the clinical diseases mentioned in this application.
Measurement of the kinase activity is a technique which is well known to the person skilled in the art. Generic test systems for the determination of the kinase activity using substrates, for example histone (for example Alessi et al., FEBS Lett. 1996, 399, 3, pages 333-338) or the basic myelin protein, are described in the literature (for example Campos-González, R. and Glenney, Jr., J. R. 1992, J. Biol. Chem. 267, page 14535).
For the identification of kinase inhibitors, various assay systems are available. In scintillation proximity assay (Sorg et al., J. of Biomolecular Screening, 2002, 7, 11-19) and flashplate assay, the radioactive phosphorylation of a protein or peptide as substrate with γATP is measured. In the presence of an inhibitory compound, a decreased radioactive signal, or none at all, is detectable. Furthermore, homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET) and fluorescence polarisation (FP) technologies are suitable as assay methods (Sills et al., J. of Biomolecular Screening, 2002, 191-214).
Other non-radioactive ELISA assay methods use specific phospho-antibodies (phospho-ABs). The phospho-AB binds only the phosphorylated substrate. This binding can be detected by chemiluminescence using a second peroxidase-conjugated anti-sheep antibody (Ross et al., 2002, Biochem. J.).
There are many diseases associated with deregulation of cellular proliferation and cell death (apoptosis). The conditions of interest include, but are not limited to, the following. The compounds according to the invention are suitable for the treatment of various conditions where there is proliferation and/or migration of smooth muscle cells and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, for example in the case of neointimal occlusive lesions. Occlusive graft vascular diseases of interest include atherosclerosis, coronary vascular disease after grafting, vein graft stenosis, peri-anastomatic prosthetic restenosis, restenosis after angioplasty or stent placement, and the like.